As the most common craniofacial birth defect (1/500 to 1/1000 live births), cleft lip with or without cleft palate (CL/P) has a considerable impact on society. Treatment is intensive and prolonged, as surgical procedures are carried out from 3-6 months of age into adolescence. Dentistry, orthodontics and speech therapy continue into adulthood. Currently, our understanding of the cellular and molecular events perturbed in CL/P is inadequate. Epithelial to Mesenchymal Transition (EMT) is a fundamental cellular behavior believed to play a critical role in palatal fusion during embryonic craniofacial development. While in the chick EMT has also been demonstrated in the fusion of the facial processes that form the upper lip, it is debated whether it mediates this process in mammals. Because mice and humans share similar morphogenetic processes during craniofacial development, the mouse is a suitable model to study craniofacial morphogenesis and its abnormalities. The Pbx family of transcription factors (TFs) play critical roles in craniofacial development. Mouse embryos deficient for Pbx1 and Pbx2 (Pbx1/Pbx2) display CL/P with 100% penetrance and offer a new model for human CL/P. Here, mouse embryos with conditional loss of Pbx1 in the cephalic epithelium on a Pbx2-deficient background will be used to establish the cellular and molecular mechanisms underlying CL/P in vivo. Preliminary data highlight that: 1) Pbx1/Pbx2 loss causes persistence of the epithelial seams at the frontonasal processes, which do not fuse and yield CL/P; 2) Cells at the seams normally exhibit the mesenchymal marker vimentin, whereas in Pbx1/Pbx2 mutants they do not; 3) Pbx1 is upregulated during TGFb-mediated EMT in an epithelial cell culture system; and 4) Pbx1 over-expression in epithelial cells gives EMT phenotypes in culture. Given these results, it is hypothesized that Pbx1 acts as a novel regulator of EMT in midface morphogenesis. To test this hypothesis, Pbx1 requirements for EMT in lip morphogenesis/fusion will be dissected in vivo at the cellular (Aim1A) and molecular level (Aim1B). The effect of Pbx1 loss on cellular morphology and identity at the epithelial seam junction will be established by Electron Microscopy, Immunofluorescence with mesenchymal and epithelial markers, and genetic fate mapping of epithelial cells in mouse embryos. It will then be determined whether Snail1, a critical effector of EMT, is an in vivo target of Pbx1 at the seam junction by Chromatin Immuno Precipitation of embryonic midfaces with Pbx1 specific antibodies. Functionality of Pbx1 binding on Snail1 expression will be established by transient transfections in cultured cells. These studies will establish whether Pbx1 is required for EMT in vivo in the embryonic midface and if it executes this program by directly targeting Snail1. In parallel, Pbx1 requirements in a cellular system of EMT will be assessed using over-expression (Aim2A) and knock-down approaches (Aim2B) to uncover whether Pbx1 is sufficient and/or necessary to induce EMT in epithelial cells. Knowledge gained thorough these studies will improve prenatal diagnostics of CL/P and drive future pharmacological and tissue engineering approaches for repair.